This application is a 371 of PCT/EP98/03761, filed Jun. 19, 1998.
The present invention relates to nanoscale particles having an iron oxide containing, ferri-, ferro- or (preferably) superparamagnetic core and at least two shells surrounding said core. Said particles may be used for medical purposes, particularly for tumor therapy by hyperthermia.
It is generally believed that in comparison to their benign counterparts, transformed cells (cancer cells) of most tumors have a higher phagocytosis activity. Reasons therefor are considered to be the invasive activity into adjacent tissues and the exocytosis of lytic enzymes associated therewith as well as a higher metabolic activity of said transformed cells. In striving for a more rapid cellular proliferation the cancer cells dedifferentiate and thereby lose part of their specifity towards normal signal transduction pathways and, generally towards transmembrane processes. Most recently such changes have been recognized by, e.g., the loss/mutation of the important cell adhesion proteins and glycoproteins of the cell surface which meanwhile are considered to represent one of the prerequisites for the uncontrolled growth of malignant cells. Thus it is known that like macrophages, cancer cells incorporate fragments of normal cells or of other cell debris and can endogenously convert them into utilizable nutrients inside lysosomes. The signal recognition by which the endocytosis process is initiated in tumor cells is still not clear on principle. However, from in vivo studies on intralesionally applied particle suspensions it can be inferred that the distribution within the tissue and the incorporation into tumor cells is mainly dependent on the size and the charge of the particles. In orientation studies the present inventors have observed that with neutrally to negatively charged particles the distribution within the tumor tissue (e.g. of the carcinoma of the mamma) into the interstitial space of the tissue (microcapillaries, septa, lobuli) is very high. If the distribution of such particles is promoted by heat the process of uniform distribution even is significantly enhanced. It is assumed that neutral to negatively charged particles interact only weakly with the extracellular receptor molecules and the glycokalyx. The explanation therefor are the much more frequently negatively charged ion channels and integral proteins on the surfaces of the cells. Almost always positively charged ions that serve either the signal transduction (e.g. in the case of Ca2+) or the maintenance of the osmotic equilibrium (e.g. Na+, K+) are imported. The rather more unspecific incorporation takes place via positively charged groups of extracellular particles since in that case the cell can import utilizable biomblecules in most cases. Furthermore biogenous sugars are also recognized and imported, said importation being less specific with tumor cells due to dedifferentiated receptor molecules. Owing to higher metabolic activity and the frequently existing deficient oxygen supply of the tumor tissues, the tumor cell must furthermore conduct an anaerobic glycolysis to a much higher extent than normal cells, which in part also results in an excessive acidification of the tumor environment due to accumulation of lactate. A further result thereof is, however, also that due to the much lower energy yield of the anaerobic metabolic pathways the tumor cells consume much more substrate of high energy content which substrate therefore has to be imported into the cells in high amounts. Since endocytosis as such also is an energy consumptive process, the tumor cell is under time pressure: although by higher importation rates more substrate is imported, also much low-grade material is taken in that affords hardly any energy yield later. Additionally, the continuous division and synthesis processes consume so much energy that in this case the usually strictly controlled intake of materials from the surrounding environment would not be sufficient and a large part of the cells would die. Therefore it makes sense that the tumor cell has a survival edge with a high unspecific intracellular intake since it can certainly gain more energy more rapidly from the digestion of xe2x80x9crawxe2x80x9d elements than in the case of the highly selective intake of fewer, more specific elements.
Through observations in cell cultures and in experimental tumors the present inventors have found that the intracellular incorporation rate of (highly) positively charged particles into tumor cells is up to 1000 times higher than that of comparable particles with neutral or negative surface charge. This is attributed to the high affinity of the positive charges of the particles towards the many negatively charged integral proteins and receptors on the cell surface. When observing a shorter period of time, e.g., 6 to 48 hours the tumor cells which are more active with respect to metabolism and division take in much higher amounts of such particles than normal cells, even with the affinity of the particles towards the surfaces of the normal cells being the same. If additionally the lower intake specifity of the tumor cells is taken into account there is a substantial overall difference in cellular intake which in theory should be exploitable for therapeutic purposes. For said purpose no systemic enrichment is necessary, but merely a skillful exploitation of surface charges for the adhesion of the particles to the cell surfaces of the tumor cells.
Particles having the highest achievable external positive charge become electrostatically bound to the cells within a few seconds and in the case of tumor cells they are also internalized within only 2 to 6 hours in such amounts that already by means of the intracellular proportion of the (nano-) particles alone compact cell pellets can be heated (to 45-47xc2x0 C.) and deactivated in vitro by an external alternating magnetic field. However, in vivo a very poor distribution of such (highly) positively charged particles within the tissue is found. In comparison thereto, neutral or negatively charged particles show a better distribution within the tissue but become less well imported into the cells and are predominantly transported away by the RES instead. Thus, for example, studies with dextran-coated magnetite particles have shown that the dextran was degraded endogenously and thereby an optimum energy intake inside the tumor cells was prevented. The present inventors have furthermore found that even though magnetite particles provided with a coating having positive charges (e.g. based on aminosilane) were not degraded endogenously they showed a poor distribution within the tissue. It would therefore be desirable to have available particles which combine the properties of the two (magnetite) particles just described, i.e., on the one hand show a very good distribution within the tumor tissue and on the other hand are also incorporated well by the tumor cells.